Pancreatic Safety of Once-Weekly Dulaglutide in Chinese Patients with Type 2 Diabetes Mellitus: Subgroup Analysis by Potential Influencing Factors.

INTRODUCTION: In the randomized, open-label, parallel-arm, active-controlled phase III AWARD-CHN2 trial, once-weekly dulaglutide plus concomitant oral antihyperglycemic medications (OAMs) improved HbA1c over 26 weeks compared with once-daily insulin glargine in patients with type 2 diabetes mellitus (T2DM). This post-hoc subgroup analysis of AWARD-CHN2 investigated the pancreatic safety of dulaglutide in Chinese patients with T2DM, stratified by potential influencing factors. METHODS: Changes in pancreatic enzyme (pancreatic amylase, total amylase, and lipase) levels over 26 weeks were assessed and stratified by patient age (< 60, ≥ 60 years), sex (female, male), duration of diabetes (< 10, ≥ 10 years), baseline weight (< 70, ≥ 70 kg), BMI (< 25, ≥ 25 kg/m2), HbA1c (< 8.5, ≥ 8.5%), triglycerides (< 2.3, ≥ 2.3 mmol/L), and concomitant OAMs (metformin, sulfonylurea, metformin plus sulfonylurea). RESULTS: A total of 203 Chinese patients with T2DM were included in this post-hoc analysis. Pancreatic enzyme levels increased within the normal range from baseline to Week 26, and no pancreatitis events were confirmed by independent adjudication. Least-squares mean increase in pancreatic amylase (U/L) from baseline to Week 26 was comparable across all subgroups with no statistically (all P-values > 0.05) or clinically significant between-group differences for age (< 60 years: 5.34; ≥ 60 years: 6.71), sex (female: 5.85; male: 5.66), duration of diabetes (< 10 years: 6.15; ≥ 10 years: 4.85), weight (< 70 kg: 6.19; ≥ 70 kg: 5.39), BMI (< 25 kg/m2: 5.92; ≥ 25 kg/m2: 5.61), HbA1c (< 8.5%: 6.82; ≥ 8.5%: 4.08), triglycerides (< 2.3 mmol/L: 4.94; ≥ 2.3 mmol/L: 8.04), and concomitant OAMs (metformin: 5.68; sulfonylurea: 5.44; metformin plus sulfonylurea: 5.87). Similar results were observed for total amylase and lipase. CONCLUSION: In Chinese patients with T2DM receiving dulaglutide 1.5 mg in AWARD-CHN2, elevations of pancreatic enzymes over 26 weeks were within the normal range and were neither associated with pancreatitis nor baseline factors, which suggests the clinical use of dulaglutide in Chinese patients with T2DM is not associated with pancreatic safety issues. CLINICAL TRIAL REGISTRATION: NCT01648582.

The Unmet Medical Needs of Current Injectable Antidiabetic Therapies in China: Patient and Health Care Professional Perspectives.

PURPOSE: Patients with diabetes and health care professionals (HCPs) play important roles in effective application of injectable antidiabetic therapies (IATs). However, their concerns and opinions on IATs are rarely investigated in China. This study aims to assess unmet medical needs of IATs regarding patient concerns, patient satisfaction, aspects that need improvement, and training burden from patient and HCP perspectives. METHODS: This cross-sectional survey was conducted in 12 representative Chinese cities from December 2018 to January 2019. Patients with adult type 2 diabetes who were receiving IAT currently and had received IAT continuously for at least 1 month before the survey, endocrinologists with ≥5 years of experience and prescribing IAT in the past 1 month, and nurses with ≥3 years of experience and providing IAT training in the past 1 month were eligible participants. The patient survey assessed concerns of initiating IAT, satisfaction with IAT, aspects of IAT that need improvement, and IAT training received. The HCP survey evaluated patient concern of initiating IAT, aspects of IAT that need improvement, experience of providing IAT training, and self-reported burden of training. Descriptive statistical analysis was performed. FINDINGS: In total, 500 patients, 200 endocrinologists, and 100 nurses were surveyed. The mean (SD) age of patients was 55.1 (11.8) years, with a disease duration of 7.6 (6.4) years. Of all patients, 391 (78.2%) were insulin users and 109 (21.8%) were glucagon-like peptide 1 receptor agonist users. Of the top 4 concerns about initiating IAT, both patients and endocrinologists reported inconvenience of daily injection (58.0% of patients and 68.5% of endocrinologists), worries about insulin dependence (42.6% of patients and 62.5% of endocrinologists), and fear of injection (37.0% of patients and 66.5% of endocrinologists). Medical expenses, convenience of drug portability and storage, and injection site reactions were the top 3 aspects that need improvement according to both patients and HCPs. High injection frequency was also one of the most urgent aspects for improvement (mean urgency score, 3.8 for physicians and 4.0 for nurses). A typical IAT training session took a mean (SD) of 14.1 (9.7) minutes. Both patients and HCPs considered injection operation after dose is set and symptoms and treatment for adverse effects as the 2 most time-consuming training contents. In addition, 97.1% of endocrinologists who provided training and 97.0% of nurses thought a more user-friendly IAT would reduce their training burden. IMPLICATIONS: Study results indicate that the IATs with more convenient drug portability and storage, fewer injection site reactions and adverse events, less injection frequency, more user-friendly design, and fewer steps for injection might help improve patient experience with self-injection and reduce HCPs' training burden.

SOCS3-deficient lung epithelial cells uptaking neutrophil-derived SOCS3 worsens lung influenza infection.

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of TBK1 and interferon pathway and the expression of SOCS3 is closely correlated with symptoms of influenza patients. However, whether deletion of Socs3 in the lung epithelial cells would affect influenza lung replication and inflammation in vivo is unknown. To test this, we approached the influenza infected Socs3f/f and SpcCre.Socs3f/f mice. We first found that knockdown of Socs3 in lung epithelial cells reduced influenza replication. However, in the in vivo study, there was a reduction of SOCS3 in the influenza-infected neutrophils coincided with an increase of SOCS3 in the CD45-CD326+ lung epithelial cells in PR8-infected SpcCre.Socs3f/f mice. SOCS3-deficient neutrophils expressed higher levels of IL-17 that enhanced chemokine expression in the lung epithelial cells. Lung SOCS3-dificient epithelial cells increased expression of GM-CSF and PGE2 which promoted SpcCre.Socs3f/f neutrophils to yield SOCS3. SpcCre.Socs3f/f lung epithelial cells internalized SOCS3 released from GM-CSF + PGE2-stimulated SpcCre.Socs3f/f neutrophils, which could boost influenza replication in the lung epithelial cells. Thus, in the in vivo study, deletion of SOCS3 from lung epithelium could be nullified by the uptake from SOCS3 from infiltrated neutrophils. In addition, deletion of Socs3 from myeloid cells reduced lung influenza infection, but increased lung inflammation. Taken together, deletion of SOCS3 could suppress influenza replication, but intracellular SOCS3 communication between neutrophils and lung epithelial cells confounds this effect.

Associations between metformin use and vitamin B12 levels, anemia, and neuropathy in patients with diabetes: a meta-analysis.

BACKGROUND: Metformin is first-line therapy for patients with diabetes. However, it may lower vitamin B12 concentrations, which could have hematological or neurological implications. This meta-analyses reviewed all available studies on associations between metformin use and vitamin B12 levels, anemia, and neuropathy in diabetic patients. METHODS: PubMed, Web of Knowledge, Cochrane Library, and Embase were searched to identify all relevant studies published in English prior to March 2018. Pooled risk ratios (RRs) and 95% confidence intervals (CIs) were calculated for dichotomous outcomes and pooled mean differences (MDs) and 95% CIs were calculated for continuous outcomes. RESULTS: Thirty-one studies were included in the meta-analyses. Compared with diabetic patients not taking metformin, patients taking metformin had a significantly higher risk of vitamin B12 deficiency (RR 2.09; 95% CI 1.49, 2.93; P < 0.0001; I2 = 64%) and significantly lower serum vitamin B12 concentrations (MD -63.70; 95% CI -74.35, -53.05] pM; P < 0.00001; I2 = 87%), which depended on dose and duration of treatment. Metformin use was also associated with significantly greater percentage decrease in serum vitamin B12 concentrations from baseline in diabetic patients (MD -14.68%; 95% CI -17.98%, -11.39%; P < 0.00001; I2 = 33%). Analyses revealed no significant association between metformin use and the prevalence of anemia or neuropathy. CONCLUSIONS: Metformin use led to significantly lowered vitamin B12 concentrations and significantly higher risk of vitamin B12 deficiency in diabetic patients. More quality studies are needed to explore the associations between metformin use and anemia and neuropathy in these patients. Annual vitamin B12 assessment in diabetic patients taking metformin is recommended.

A novel role of endocan in alleviating LPS-induced acute lung injury.

AIMS: Endotoxin induced acute lung injury (ALI) is a critical complication of some clinical illnesses. Endothelial cell dysfunction and excessive pro-inflammation cytokine release are pivotal to the injury of alveolar-capillary membrane which is the typical characteristic of endotoxic lung injury. As a potential marker of endothelial cells, endocan plays an important role in many endothelial-dependent pathophysiological diseases. We speculated that endocan have anti-inflammatory property in ALI. Here, we investigated the role of endocan in LPS-induced ALI. MATERIALS AND METHODS: Mice were randomly divided into 4 groups. LPS were used to construct ALI mice model by aerosolization for 20 min. Endocan was intraperitoneal injected at 30 min before LPS exposure. Levels of TNF-α, IFN-γ, IL-1ß, IL-6 and MPO activities were detected by indicated ELISA. Cell apoptotic rate was determined by Annexin V/PI kit, ROS level and MPTP were detected by DCFH-DA and JC-1 kit, respectively. Seahorse XF96 was applied to evaluate the alteration of OCR and ECAR. Western blot and qRT-PCR were used to detect indicated molecules. KEY FINDINGS: Endocan effectively decreased TNF-α, IFN-γ, IL-1ß, and IL-6 levels as well as relieved pulmonary epithelium cell apoptosis caused by LPS exposure. Endocan significantly reversed LPS induced UPRmt and promoted cell metabolism reprogramming which were crucial for the protective characteristic of endocan in ALI mice model. SIGNIFICANCE: The above findings suggested endocan could significantly suppress inflammatory response in ALI model through attenuating UPRmt associated apoptosis and switch cellular bioenergetics, indicating endocan could be considered as a promising compound against LPS induced ALI.

Determination of corypalmine in mouse blood by UPLC-MS/MS and its application to a pharmacokinetic study.

In this work, a selective and sensitive ultra-performance liquid chromatography tandem mass spectrometry method was established and validated for determination of corypalmine in mouse blood after oral or intravenous administration. A UPLC BEH C18 column was used to separate corypalmine and berberrubine (internal standard) at 40°C. The mobile phase was composed of acetonitrile and 10 mmol/L ammonium acetate (containing 0.1% formic acid) at a flow rate of 0.4 mL/min, and the total run time was 4.0 min. Electrospray ionization in positive ion mode was applied; target fragment ions m/z 342.2 → 178.0 for corypalmine and m/z 322.1 → 307.0 for berberrubine were identified with multiple reaction monitoring mode. The linear range was 1-1000 ng/mL (r > 0.995) and the lower limit of quantification for corypalmine in plasma was 1.0 ng/mL. The intra- and inter-day precisions were both <14%. The range of accuracy in this method was 97.5-109.0%. Mean recovery was >69.6%, and the matrix effect was 96.8-107.6%. Based on its high sensitivity, specificity and reliability, this method was successfully applied to study the pharmacokinetic parameters of corypalmine in mouse by oral and intravenous administration, and finally, the bioavailability of corypalmine was identified at 4.6%.

Pharmacist-driven antimicrobial stewardship in intensive care units in East China: A multicenter prospective cohort study.

BACKGROUND: Antimicrobial stewardship programs, particularly pharmacist-driven programs, help reduce the unnecessary use of antimicrobial agents. The objective of this study was to assess the influence of pharmacist-driven antimicrobial stewardship on antimicrobial use, multidrug resistance, and patient outcomes in adult intensive care units in China. METHOD: We conducted a multicenter prospective cohort study with a sample of 577 patients. A total of 353 patients were included under a pharmacist-driven antimicrobial stewardship program, whereas the remaining 224 patients served as controls. The primary outcome was all-cause hospital mortality. RESULTS: The pharmacist-driven antimicrobial stewardship program had a lower hospital mortality rate compared with the nonpharmacist program (19.3% vs 29.0%; P = .007). Furthermore, logistic regression analysis indicated that the pharmacist-driven program independently predicted hospital mortality (odds ratio, 0.57; 95% confidence interval, 0.36-0.91; P = .017) after adjustment. Meanwhile, this strategy had a lower rate of multidrug resistance (23.8% vs 31.7%; P = .037). Moreover, the strategy optimized antimicrobial use, such as having a shorter duration of empirical antimicrobial therapy (2.7 days; interquartile range [IQR], 1.7-4.6 vs 3.0; IQR, 1.9-6.2; P = .002) and accumulated duration of antimicrobial treatment (4.0; IQR, 2.0-7.0 vs 5.0; IQR, 3.0-9.5; P = .030). CONCLUSIONS: Pharmacist-driven antimicrobial stewardship in an intensive care unit decreased patient mortality and the emergence of multidrug resistance, and optimized antimicrobial agent use.

A multifactor model for predicting mortality in critically ill patients: A multicenter prospective cohort study.

PURPOSE: The objective of this study was to develop a model using a combination of routine clinical variables to predict mortality in critically ill patients. METHODS: A cohort of 500 patients recruited from eight university hospital intensive care units (ICUs) was used to develop a model via logistic regression analyses. Discrimination and calibration analyses were performed to assess the model. RESULTS: The model included the lactate level (odds ratio [OR]=1.11, 95% confidence interval [CI] 1.01 to 1.22, P=0.029), neutrophil-to-lymphocyte ratio (OR=1.03, 95% CI 1.01 to 1.04, P=0.002), acute physiology score (OR=1.11, 95% CI 1.06 to 1.15, P<0.001), Charlson comorbidity index (OR=1.36, 95% CI 1.15 to 1.60, P<0.001) and surgery type (OR: selective=Ref, no surgery=8.04, 95% CI 3.74 to 17.30, P<0.001, emergency=3.66, 95% CI 1.60 to 8.36, P=0.002). The model showed good discrimination (area under receiver operating characteristic curve: 0.84, 95% CI: 0.80 to 0.87) and calibration (Hosmer-Lemeshow test P=0.137) for predicting in-hospital mortality. CONCLUSION: The developed multifactor model can be used to effectively predict mortality in critically ill patients at ICU admission.

Insulin adherence and persistence among Chinese patients with type 2 diabetes: a retrospective database analysis.

OBJECTIVE: To assess adherence and persistence to insulin therapy and identify its associated factors among Chinese insulin-naïve patients with type 2 diabetes (T2D). METHODS: Tianjin Urban Employee Basic Medical Insurance claims database was used (2008-2011). Adult patients with T2D who initiated insulin therapy during January 2009 through December 2010 and were continuously enrolled for 12 months pre-(baseline) and 12 months post-initiation (follow-up) were included. Patients who had a ≥80% medication possession ratio were deemed adherent, while patients who had no gaps of ≥90 days in insulin therapy were deemed persistent. Associated factors of insulin adherence and persistence were detected by univariate and multivariate analyses. RESULTS: A total of 24,192 patients were included; the patients had a mean age of 58.9 years, with 49.5% being female. About 51.9% of the patients had human insulin as initiation therapy, while 39.1% were initiated with insulin analog and 9.0% with animal-derived insulin. Premixed insulin (77.3%) was prescribed most often in comparison with basal (11.8%) and prandial (10.9%) insulin. Only 30.9% of patients were adherent, and the mean (standard deviation) medication possession ratio was 0.499 (0.361). About 53.0% of patients persisted insulin therapy during follow-up, and the mean time to nonpersistence was 230.3 (145.5) days. Patients initiated with analog were more likely to be adherent (adjusted odds ratio: 1.07, P=0.036) and persistent (adjusted hazard ratio: 0.88, P<0.001) compared with those initiated with human insulin. Patients initiation with basal insulin had lower adherence relative to premixed (adjusted odds ratio: 0.79, P<0.001). Patients comorbid with hypertension or dyslipidemia, initiated with prandial insulin, and with baseline severe hypoglycemic events were more likely to be nonadherent/nonpersistent. CONCLUSION: The insulin adherence and persistence among Chinese patients with T2D are generally poor. Initiation with insulin analog or premixed insulin may result in better adherence/persistence to insulin therapy.

Pharmacokinetic study of dendrobine in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry.

Dendrobine, considered as the major active alkaloid compound, has been used for the quality control and discrimination of Dendrobium which is documented in the Chinese Pharmacopoeia. In this work, a sensitive and simple ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for determination of dendrobine in rat plasma is developed. After addition of caulophyline as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 (2.1 ×100 mm, 1.7 µm) column with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 264.2 → 70.0 for dendrobine and m/z 205.1 → 58.0 for IS. Calibration plots were linear throughout the range 2-1000 ng/mL for dendrobine in rat plasma. The RSDs of intra-day and inter-day precision were both <13%. The accuracy of the method was between 95.4 and 103.9%. The method was successfully applied to pharmacokinetic study of dendrobine after intravenous administration. Copyright © 2015 John Wiley & Sons, Ltd.

Metabolic changes in rats after intragastric administration of MGCD0103 (Mocetinostat), a HDAC class I inhibitor.

MGCD0103, an isotype-selective HDACi, has been clinically evaluated for the treatment of hematologic malignancies and advanced solid tumors, alone and in combination with standard-of-care agents. In this study, we developed a serum metabolomic method based on gas chromatography-mass spectrometry (GC-MS) to evaluate the effect of intragastric administration of MGCD0103 on rats. The MGCD0103 group rats were given 20, 40, 80 mg/kg of MGCD0103 by intragastric administration each day for 7 days. Pattern recognition analysis, including both principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) revealed that intragastric administration of MGCD0103 induced metabolic perturbations. As compared to the control group, the levels of L-alanine, L-isoleucine, and L-leucine of MGCD0103 group decreased. The results indicate that metabolomic methods based on GC-MS may be useful to elucidate side effect of MGCD0103 through the exploration of biomarkers (L-alanine, L-isoleucine, and L-leucine). According to the pathological changes of liver at difference dosage, MGCD0103 is hepatotoxic and its toxity is dose-dependent.

Determination of N-methylcytisine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study.

In this work, a sensitive and selective UPLC-MS/MS method for determination of N-methylcytisine in rat plasma is developed. After addition of hordenine as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm×100mm, 1.7µm) with acetonitrile (containing 10mM ammonium formate) and water (containing 0.1% formic acid and 10mM ammonium formate) as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 205.1→58.0 for N-methylcytisine, and m/z 166.1→121.0 for IS. Calibration plots were linear throughout the range 2-2000ng/mL for N-methylcytisine in rat plasma. Mean recoveries of N-methylcytisine in rat plasma ranged from 86.1% to 94.8%. RSD of intra-day and inter-day precision were both<13%. The accuracy of the method was between 94.5% and 109.4%. The method was successfully applied to pharmacokinetic study of N-methylcytisine after either oral or intravenous administration. For the first time, the absolute bioavailability of N-methylcytisine was reported as high as 55.5%.

Roxithromycin suppresses airway remodeling and modulates the expression of caveolin-1 and phospho-p42/p44MAPK in asthmatic rats.

Roxithromycin (RXM) expresses anti-asthmatic effects that are separate from its antibiotic activity, but its effects on airway remodeling are still unknown. Here, we evaluated the effects of RXM on airway remodeling and the expression of caveolin-1 and phospho-p42/p44mitogen-activated protein kinase (phospho-p42/p44MAPK) in chronic asthmatic rats. The chronic asthma was induced by ovalbumin/Al(OH)3 sensitization and ovalbumin challenge, RXM (30mg/kg) or dexamethasone (0.5mg/kg) was given before airway challenge initiation. We measured the thickness of bronchial wall and bronchial smooth muscle cell layer to indicate airway remodeling, and caveolin-1 and phospho-p42/p44MAPK expression in lung tissue and airway smooth muscle were detected by immunohistochemistry and western blot analysis, respectively. The results demonstrated that RXM treatment decreased the thickness of bronchial wall and bronchial smooth muscle cell layer, and also downregulated the phospho-p42/p44MAPK expression and upregulated the caveolin-1 expression. The above effects of RXM were similar to dexamethasone. Our results suggested that pretreatment with RXM could suppress airway remodeling and regulate the expression of caveolin-1 and phospho-p42/p44MAPK in chronic asthmatic rats.

Vagus nerve through α7 nAChR modulates lung infection and inflammation: models, cells, and signals.

Cholinergic anti-inflammatory pathway (CAP) bridges immune and nervous systems and plays pleiotropic roles in modulating inflammation in animal models by targeting different immune, proinflammatory, epithelial, endothelial, stem, and progenitor cells and signaling pathways. Acute lung injury (ALI) is a devastating inflammatory disease. It is pathogenically heterogeneous and involves many cells and signaling pathways. Here, we emphasized the research regarding the modulatory effects of CAP on animal models, cell population, and signaling pathways that involved in the pathogenesis of ALI. By comparing the differential effects of CAP on systemic and pulmonary inflammation, we postulated that a pulmonary parasympathetic inflammatory reflex is formed to sense and respond to pathogens in the lung. Work targeting the formation and function of pulmonary parasympathetic inflammatory reflex would extend our understanding of how vagus nerve senses, recognizes, and fights with pathogens and inflammatory responses.

Lipoxin A4 and platelet activating factor are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice.

CFTR (cystic fibrosis transmembrane conductance regulator) is expressed by both neutrophils and platelets. Lack of functional CFTR could lead to severe lung infection and inflammation. Here, we found that mutation of CFTR (F508del) or inhibition of CFTR in mice led to more severe thrombocytopenia, alveolar neutrocytosis and bacteriosis, and lower lipoxin A4/MIP-2 (macrophage inhibitory protein-2) or lipoxin A4/neutrophil ratios in the BAL (bronchoalveolar lavage) during acute E. coli pneumonia. In vitro, inhibition of CFTR promotes MIP-2 production in LPS-stimulated neutrophils; however, lipoxin A4 could dose-dependently suppress this effect. In LPS-induced acute lung inflammation, blockade of PSGL-1 (P-selectin glycoprotein ligand-1) or P-selectin, antagonism of PAF by WEB2086, or correction of mutated CFTR trafficking by KM11060 could significantly increase plasma lipoxin A4 levels in F508del relevant to wildtype mice. Concurrently, F508del mice had higher plasma platelet activating factor (PAF) levels and PAF-AH activity compared to wildtype under LPS challenge. Inhibiting hydrolysis of PAF by a specific PAF-AH (PAF-acetylhydrolase) inhibitor, MAFP, could worsen LPS-induced lung inflammation in F508del mice compared to vehicle treated F508del group. Particularly, depletion of platelets in F508del mice could significantly decrease plasma lipoxin A4 and PAF-AH activity and deteriorate LPS-induced lung inflammation compared to control F508del mice. Taken together, lipoxin A4 and PAF are involved in E. coli or LPS-induced lung inflammation in CFTR-deficient mice, suggesting that lipoxin A4 and PAF might be therapeutic targets for ameliorating CFTR-deficiency deteriorated lung inflammation.

Differential regulation of mouse pancreatic islet insulin secretion and Smad proteins by activin ligands.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is regulated by paracrine factors, the identity and mechanisms of action of which are incompletely understood. Activins are expressed in pancreatic islets and have been implicated in the regulation of GSIS. Activins A and B signal through a common set of intracellular components, but it is unclear whether they display similar or distinct functions in glucose homeostasis. METHODS: We examined glucose homeostatic responses in mice lacking activin B and in pancreatic islets derived from these mutants. We compared the ability of activins A and B to regulate downstream signalling, ATP production and GSIS in islets and beta cells. RESULTS: Mice lacking activin B displayed elevated serum insulin levels and GSIS. Injection of a soluble activin B antagonist phenocopied these changes in wild-type mice. Isolated pancreatic islets from mutant mice showed enhanced GSIS, which could be rescued by exogenous activin B. Activin B negatively regulated GSIS and ATP production in wild-type islets, while activin A displayed the opposite effects. The downstream mediator Smad3 responded preferentially to activin B in pancreatic islets and beta cells, while Smad2 showed a preference for activin A, indicating distinct signalling effects of the two activins. In line with this, overexpression of Smad3, but not Smad2, decreased GSIS in pancreatic islets. CONCLUSIONS/INTERPRETATION: These results reveal a tug-of-war between activin ligands in the regulation of insulin secretion by beta cells, and suggest that manipulation of activin signalling could be a useful strategy for the control of glucose homeostasis in diabetes and metabolic disease.

Important role of platelets in modulating endotoxin-induced lung inflammation in CFTR-deficient mice.

Mutation of CFTR (cystic fibrosis transmembrane conductance regulator) leads to cystic fibrosis (CF). Patients with CF develop abnormalities of blood platelets and recurrent lung inflammation. However, whether CFTR-mutated platelets play a role in the development of lung inflammation is elusive. Therefore, we intratracheally challenged wildtype and F508del (a common type of CFTR mutation) mice with LPS to observe changes of F508del platelets in the peripheral blood and indexes of lung inflammation (BAL neutrophils and protein levels). Furthermore, we investigated whether or not and how F508del platelets modulate the LPS-induced acute lung inflammation by targeting anti-platelet aggregation, depletion of neutrophils, reconstitution of bone marrow or neutrophils, blockade of P-selectin glycoprotein ligand-1 (PSGL-1), platelet activating factor (PAF), and correction of mutated CFTR trafficking. We found that LPS-challenged F508del mice developed severe thrombocytopenia and had higher levels of plasma TXB2 coincided with neutrophilic lung inflammation relative to wildtype control. Inhibition of F508del platelet aggregation or depletion of F508del neutrophils diminished the LPS-induced lung inflammation in the F508del mice. Moreover, wildtype mice reconstituted with either F508del bone marrow or neutrophils developed worse thrombocytopenia. Blocking PSGL-1, platelet activating factor (PAF), or rectifying trafficking of mutated CFTR in F508del mice diminished and alveolar neutrophil transmigration in the LPS-challenged F508del mice. These findings suggest that F508del platelets and their interaction with neutrophils are requisite for the development of LPS-induced lung inflammation and injury. As such, targeting platelets might be an emerging strategy for dampening recurrent lung inflammation in cystic fibrosis patients.

Neuroendocrine control of female reproductive function by the activin receptor ALK7.

Activins are critical components of the signaling network that controls female reproduction. However, their roles in hypothalamus, and the specific functions of their different receptors, have not been elucidated. Here, we investigated the expression and function of the activin receptor ALK7 in the female reproductive axis using Alk7-knockout mice. ALK7 was found in subsets of SF1-expressing granulosa cells in the ovary, FSH gonadotrophs in the pituitary, and NPY-expressing neurons in the arcuate nucleus of the hypothalamus. Alk7-knockout females showed delayed onset of puberty and abnormal estrous cyclicity, had abnormal diestrous levels of FSH and LH in serum, and their ovaries showed premature depletion of follicles, oocyte degeneration, and impaired responses to exogenous gonadotropins. In the arcuate nucleus, mutant mice showed reduced expression of Npy mRNA and lower numbers of Npy-expressing neurons than wild-type controls. Alk7 knockouts showed a selective loss of arcuate NPY/AgRP innervation in the medial preoptic area, a key central regulator of reproduction. These results indicate that ALK7 is an important regulator of female reproductive function and reveal a new role for activin signaling in the control of hypothalamic gene expression and wiring. Alk7 gene variants may contribute to female reproductive disorders in humans, such as polycystic ovary syndrome.

Determination of pethidine in human plasma by LC-MS/MS.

A sensitive and selective liquid chromatography-tandem mass spectrometry method for the determination of pethidine in human plasma was developed and validated over the concentration range of 4-2000 ng/mL. After addition of ketamine as internal standard, liquid-liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 100 × 2.1 mm, 5 µm particle, Allure™ PFP propyl column, with 45:40:15 (v/v/v) acetonitrile-methanol-water containing 0.2% formic acid as mobile phase. The MS data acquisition was accomplished by multiple reactions monitoring mode with positive electrospray ionization interface. The lower limit of quantification was 4 ng/mL; for inter-day and intra-day tests, the precision (RSD) for the entire validation was less than 7%, and the accuracy was within 95.9-106.5%. The method is sensitive and simple, and was successfully applied to analysis of samples of clinical intoxication.

Sustained viral load and late death in Rag2-/- mice after influenza A virus infection.

The importance of the adaptive immune response for secondary influenza infections and protection from a lethal challenge after vaccination has been well documented. However, some controversy still exists concerning the specific involvement of B and T cells during a primary infection. Here, we have followed the survival, weight loss, viral load and lung pathology in Rag2-/- knock-out mice after infection with influenza A virus (H1N1). Infected wild type mice initially lost weight early after infection but then cleared the virus and recovered. Rag2-/- mice, however, showed similar weight loss kinetics in the early stages after infection but weight loss continued post infection and culminated in death. In contrast to wild type mice, Rag2-/- mice were not able to clear the virus, despite an increased inflammatory response. Furthermore, they did not recruit virus-specific lymphocytes into the lung in the later stages after infection and exhibited sustained pulmonary lesions.